Table 5 |
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Primers used for GUSB analysis |
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a) Primers used for primerwalking |
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|
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|
Primer name |
Sequence of forward primer (5'–3') |
Strand orientation |
Location in GUSB |
Template DNA |
TAnn(°C) |
|
|
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|
GAPIntron10.f |
ggggctgagtgttagacaatgca |
plus |
Intron 10 |
PAC clone |
53 |
|
GAP5prime.f |
cattaacggaagattgctttggt |
plus |
Intron 1 |
PAC clone |
53 |
|
3-Strich.f |
gcgagagaggtactggaaact |
plus |
Exon12 |
PAC clone |
50 |
|
|
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b) Primers used for long range PCR |
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|
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|
Primer name |
Sequence of primer (5'–3') |
Sequence of reverse primer (5'–3') |
Amplified part of gene |
Length of product |
TAnn(°C) |
|
|
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|
AuEx8.f/AuEx9.r |
caggctgcttactacttcaa |
ggtcacgaaggtcacg |
Intron 8 |
2660 |
55 |
|
AuEx9.f/AuEx10.r |
cgtgaccttcgtgacc |
accaggagtagtaactgttca |
Intron 9 |
1717 |
55 |
|
AuEx10.f/AuEx11.r |
atccagagcgagtacgg |
gatactgctgtagcaggc |
Intron 10 |
3221 |
55 |
|
AuEx11.f/AuEx12.r |
tatgtggttggagagctca |
caacaggaatgctgcact |
Intron 11 |
1843 |
55 |
|
|
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|
c) Primers used for SNP discovery and SNP typing |
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|
|
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|
Primer names |
Sequence of forward primer (5'–3') |
Sequence of reverse primer (5'–3') |
Amplified Exon (Length of Exon) |
Length of Product (bp) |
TAnn(°C) |
|
|
|||||
|
GUSBEx1.f/GUSBEx1.r |
atcgccttcagtcaagatgg |
ctctgggccagcagttcc |
Ex 1 (213 bp) |
455 |
60 |
|
GUSBEx2.f/GUSBEx2.r |
ggtgggtggcctggaatctgg |
gcctcccccacccctcccta |
Ex 2 (186 bp) |
465 |
60 |
|
GUSBEx3.f/GUSBEx3.r |
ttgggagggttgcctcatctct |
gagctcagattacatgcctccc |
Ex 3 (188 bp) |
465 |
65.5 |
|
GUSBEx4.f/GUSBEx4.r |
tgccgccagggaccattctc |
ggtgccggcagagcccttct |
Ex 4 (146 bp) |
458 |
59 |
|
GUSBEx5.f/GUSBEx5.r |
tcctcagagtccaggtgctcc |
acctgtccggcacccctgct |
Ex 5 (191 bp) |
358 |
65 |
|
GUSBEx6.f/GUSBEx6.r |
gtgaagctcacggcacagac |
tgctctgagctgccctcagcc |
Ex 6 (157 bp) |
241 |
65 |
|
GUSBEx7.f/GUSBEx7.r |
agcacatcttggacagggaca |
gagagggagggagtcacat |
Ex 7 (187 bp) |
295 |
65 |
|
GUSBEx8.f/GUSBEx8.r |
gatgtgactccctccctctc |
tgaggtcccatggctggctga |
Ex 8 (155 bp) |
235 |
63 |
|
GUSBEx9.F/GUSBEx9.r |
ccccaccccatcccttcctg |
gggtggaaacccggctgctt |
Ex 9 (89 bp) |
510 |
59 |
|
GUSBEx10.f/GUSBEx10.r |
ggtaggggtgccagcccaga |
gcccaggcagggagtgaca |
Ex 10 (185 bp) |
430 |
59 |
|
GUSBEx11.f/GUSBEx11.r |
gctacgtcataccttggctgtg |
ccgatccgattagcttgccct |
Ex 11 (140 bp) |
431 |
65 |
|
GUSBEx12.f/GUSBEx12.r |
cagcacgtctaaaccgtaagagga |
actaccagattcagaccagaaact |
Ex 12 (147 bp) |
409 |
60.9 |
|
|
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|
d) Primers for tetra-primer ARMS-PCR |
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|
|
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|
Primer combination |
Sequence of forward primer (5'–3') |
Sequence of reverse primer (5'–3') |
Length of product (bp) |
Detected allele |
TAnn(°C) |
|
|
|||||
|
EX5outer.f/EX5outer.r |
gcaaccgtagctcgggacacctgaga |
aagaactttcacccatcctgcccctgg |
606 |
none |
63.2 |
|
EX5innerG/EX5outer.r |
ttctggatgaggaaggcagggggg |
aagaactttcacccatcctgcccctgg |
342 |
G |
63.2 |
|
EX5outer.f/EX5innerA |
gcaaccgtagctcgggacacctgaga |
ccccccgtccccttggcaat |
308 |
A |
63.2 |
|
|
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|
Beck et al. BMC Veterinary Research 2006 2:14 doi:10.1186/1746-6148-2-14 |
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